We observed the emission of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) when excited with the fundamental output of the fs Ti:Sapphire laser at 860 nm. The emission spectra of DPH was identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotations correlation time for two- and three-photon excitation. Steady state anisotropy data for DPH labeled membranes revealed the same temperature dependence for one- and three-photon excitation. These anisotropy data indicate that sample heating was not significant with three-photon excitation, and that three-photon may thus be of practical usefulness in fluorescence spectroscopy and microscopy.